Monocytes and macrophages sense the presence of pathogens, tissue damage, and host-derived mediators in their environment and respond by differentiating into distinct functional phenotypes that mediate host innate immune responses. The process of macrophage differentiation has often been described in terms of plasticity, implying that these cells modulate their functions through rapidly reversible differentiation steps in response to environmental changes in the host. For example, classically activated macrophages (M1) are highly microbicidal, yet their production of inflammatory cytokines and nitric oxide may also damage host tissue. At the other end of the functional spectrum, alternatively activated macrophages (AA-Mf or M2), induced by IL-4 and IL- 13, mediate wound healing through elimination of damaged tissue and other anti-inflammatory mechanisms. During the past 32 years of this award, the PI undertook research designed to dissect the complex molecular underpinnings of macrophage differentiation and how this impacts host defenses and disease outcome. Work completed during the current funding cycle has focused primarily on key mechanisms by which Toll-like receptor (TLR) signaling induced by conserved microbial structures, such as bacterial LPS or Respiratory Syncytial Virus (RSV) fusion (F) protein, and cell-derived cytokines, such as interferons (IFNs), drive this complex process. In the proposed studies, the overarching hypothesis is that activation of specific intracellular signaling pathways distal to TLR and/or cytokine receptor engagement serve to program expression of discrete cassettes of pro- and anti-inflammatory genes that then dictate the manner in which the host responds to infection. Three Specific Aims are proposed to test this hypothesis in vitro and in vivo, with the ultimate goal of identifying novel therapeutic interventions for diseases where macrophages are required for containing the invading pathogen and/or resolving tissue damage caused by pathogens. Both mice (because of the availability of genetically engineered strains), and cotton rats (that are unique in their susceptibility to human non-adapted isolates of influenza and RSV) will be utilized. The proposed innovative experimental approaches are designed to: (1) identify heterologous signaling receptors with which TLR4 interacts physically and/or functionally and consequences of those interactions in regulation of macrophage responsiveness; (2) determine mechanism(s) of in- creased host susceptibility to Gram positive pathogens after influenza infection, and (3) develop an effective therapy for RSV infection based on strong preliminary data showing that TLR4-induced PPAR is central for AA-Mf differentiation, but that TLR4 also contributes to DAMP-mediated signaling. At the conclusion of these comprehensive studies, key processes that govern interactions of TLR agonists and/or IFN that lead to changes in macrophage activation will have been defined that, in turn, can be expected to translate into reasonable and practical therapeutic approaches for controlling invading pathogens or counteracting inflammatory damage to tissues induced by pathogens, ultimately providing evidence-based therapies to treat infectious diseases.